protein kinase A Dotaz Zobrazit nápovědu
BACKGROUND: Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a member of the Ca2+/calmodulin-dependent kinase (CaMK) family involved in adiposity regulation, glucose homeostasis and cancer. This upstream activator of CaMKI, CaMKIV and AMP-activated protein kinase is inhibited by phosphorylation, which also triggers an association with the scaffolding protein 14-3-3. However, the role of 14-3-3 in the regulation of CaMKK2 remains unknown. METHODS: The interaction between phosphorylated CaMKK2 and the 14-3-3γ protein, as well as the architecture of their complex, were studied using enzyme activity measurements, small-angle x-ray scattering (SAXS), time-resolved fluorescence spectroscopy and protein crystallography. RESULTS: Our data suggest that the 14-3-3 protein binding does not inhibit the catalytic activity of phosphorylated CaMKK2 but rather slows down its dephosphorylation. Structural analysis indicated that the complex is flexible and that CaMKK2 is located outside the phosphopeptide-binding central channel of the 14-3-3γ dimer. Furthermore, 14-3-3γ appears to interact with and affect the structure of several regions of CaMKK2 outside the 14-3-3 binding motifs. In addition, the structural basis of interactions between 14-3-3 and the 14-3-3 binding motifs of CaMKK2 were elucidated by determining the crystal structures of phosphopeptides containing these motifs bound to 14-3-3. CONCLUSIONS: 14-3-3γ protein directly interacts with the kinase domain of CaMKK2 and the region containing the inhibitory phosphorylation site Thr145 within the N-terminal extension. GENERAL SIGNIFICANCE: Our results suggested that CaMKK isoforms differ in their 14-3-3-mediated regulations and that the interaction between 14-3-3 protein and the N-terminal 14-3-3-binding motif of CaMKK2 might be stabilized by small-molecule compounds.
- Klíčová slova
- 14-3-3 protein, CaMKK, Fluorescence spectroscopy, Protein-protein interaction, SAXS,
- MeSH
- aminokyselinové motivy MeSH
- fosforylace účinky léků MeSH
- kinasa proteinkinasy závislé na vápníku a kalmodulinu metabolismus MeSH
- konformace proteinů účinky léků MeSH
- lidé MeSH
- mapování interakce mezi proteiny MeSH
- molekulární modely MeSH
- posttranslační úpravy proteinů MeSH
- proteinkinasa závislá na vápníku a kalmodulinu typ 1 metabolismus MeSH
- proteinkinasy aktivované AMP metabolismus MeSH
- proteinové domény MeSH
- proteiny 14-3-3 metabolismus MeSH
- rekombinantní proteiny metabolismus MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- CAMK1 protein, human MeSH Prohlížeč
- CAMKK2 protein, human MeSH Prohlížeč
- kinasa proteinkinasy závislé na vápníku a kalmodulinu MeSH
- PRKAA2 protein, human MeSH Prohlížeč
- proteinkinasa závislá na vápníku a kalmodulinu typ 1 MeSH
- proteinkinasy aktivované AMP MeSH
- proteiny 14-3-3 MeSH
- rekombinantní proteiny MeSH
p53 is one of the most important regulators of cell proliferation and differentiation and of programmed cell death, triggering growth arrest and/or apoptosis in response to different cellular stress signals. The sequence-specific DNA-binding function of p53 protein can be activated by several different stimuli that modulate the C-terminal domain of this protein. The predominant mechanism of activation of p53 sequence-specific DNA binding is phosphorylation at specific sites. For example, phosphorylation of p53 by PKC (protein kinase C) occurs in undamaged cells, resulting in masking of the epitope recognized by monoclonal antibody PAb421, and presumably promotes steady-state levels of p53 activity in cycling cells. In contrast, phosphorylation by cdk2 (cyclin-dependent kinase 2)/cyclin A and by the protein kinase CK2 are both enhanced in DNA-damaged cells. We determined whether one mechanism to account for this mutually exclusive phosphorylation may be that each phosphorylation event prevents modification by the other kinase. We used non-radioactive electrophoretic mobility shift assays to show that C-terminal phosphorylation of p53 protein by cdk2/cyclin A on Ser315 or by PKC on Ser378 can efficiently stimulate p53 binding to DNA in vitro, as well as binding of the monoclonal antibody Bp53-10, which recognizes residues 371-380 in the C-terminus of p53. Phosphorylation of p53 by CK2 on Ser392 induces its DNA-binding activity to a much lower extent than phosphorylation by cdk2/cyclin A or PKC. In addition, phosphorylation by CK2 strongly inhibits PKC-induced activation of p53 DNA binding, while the activation of p53 by cdk2/cyclin A is not affected by CK2. The presence of CK2-mediated phosphorylation promotes PKC binding to its docking site within the p53 oligomerization domain, but decreases phosphorylation by PKC, suggesting that competition between CK2 and PKC does not rely on the inhibition of PKC-p53 complex formation. These results indicate the crucial role of p53 C-terminal phosphorylation in the regulation of its DNA-binding activity, but also suggest that antagonistic relationships exist between different stress signalling pathways.
- MeSH
- buněčné linie MeSH
- cyklin-dependentní kinasa 2 MeSH
- DNA chemie metabolismus MeSH
- fosforylace MeSH
- kaseinkinasa II MeSH
- kinasy CDC2-CDC28 metabolismus MeSH
- lidé MeSH
- monoklonální protilátky farmakologie MeSH
- nádorový supresorový protein p53 chemie imunologie metabolismus MeSH
- protein-serin-threoninkinasy metabolismus MeSH
- proteinkinasa C antagonisté a inhibitory metabolismus MeSH
- responzivní elementy MeSH
- Spodoptera cytologie MeSH
- terciární struktura proteinů MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- CDK2 protein, human MeSH Prohlížeč
- cyklin-dependentní kinasa 2 MeSH
- DNA MeSH
- kaseinkinasa II MeSH
- kinasy CDC2-CDC28 MeSH
- monoklonální protilátky MeSH
- nádorový supresorový protein p53 MeSH
- protein-serin-threoninkinasy MeSH
- proteinkinasa C MeSH
Phosphoinositide 3-kinases (PI3K) and phosphoinositide 3-kinase-related protein kinases (PIKK) are two structurally related families of kinases that play vital roles in cell growth and DNA damage repair. Dysfunction of PIKK members and aberrant stimulation of the PI3K/AKT/mTOR signalling pathway are linked to a plethora of diseases including cancer. In recent decades, numerous inhibitors related to the PI3K/AKT/mTOR signalling have made great strides in cancer treatment, like copanlisib and sirolimus. Notably, most of the PIKK inhibitors (such as VX-970 and M3814) related to DNA damage response have also shown good efficacy in clinical trials. However, these drugs still require a suitable combination therapy to overcome drug resistance or improve antitumor activity. Based on the aforementioned facts, we summarised the efficacy of PIKK, PI3K, and AKT inhibitors in the therapy of human malignancies and the resistance mechanisms of targeted therapy, in order to provide deeper insights into cancer treatment.
- Klíčová slova
- AKT, PI3K, PIKK, anticancer therapy, inhibitors,
- MeSH
- 1-fosfatidylinositol-3-kinasa * metabolismus terapeutické užití MeSH
- fosfatidylinositol-3-kinasy metabolismus MeSH
- inhibitory fosfoinositid-3-kinasy farmakologie MeSH
- inhibitory proteinkinas farmakologie terapeutické užití MeSH
- lidé MeSH
- nádory * farmakoterapie MeSH
- protoonkogenní proteiny c-akt metabolismus MeSH
- TOR serin-threoninkinasy metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- 1-fosfatidylinositol-3-kinasa * MeSH
- fosfatidylinositol-3-kinasy MeSH
- inhibitory fosfoinositid-3-kinasy MeSH
- inhibitory proteinkinas MeSH
- protoonkogenní proteiny c-akt MeSH
- TOR serin-threoninkinasy MeSH
Apoptosis signal-regulating kinase 1 (ASK1, also known as MAP3K5), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, regulates diverse physiological processes. The activity of ASK1 is triggered by various stress stimuli and is involved in the pathogenesis of cancer, neurodegeneration, inflammation, and diabetes. ASK1 forms a high molecular mass complex whose activity is, under non-stress conditions, suppressed through interaction with thioredoxin and the scaffolding protein 14-3-3. The 14-3-3 protein binds to the phosphorylated Ser-966 motif downstream of the ASK1 kinase domain. The role of 14-3-3 in the inhibition of ASK1 has yet to be elucidated. In this study we performed structural analysis of the complex between the ASK1 kinase domain phosphorylated at Ser-966 (pASK1-CD) and the 14-3-3ζ protein. Small angle x-ray scattering (SAXS) measurements and chemical cross-linking revealed that the pASK1-CD·14-3-3ζ complex is dynamic and conformationally heterogeneous. In addition, structural analysis coupled with the results of phosphorus NMR and time-resolved tryptophan fluorescence measurements suggest that 14-3-3ζ interacts with the kinase domain of ASK1 in close proximity to its active site, thus indicating this interaction might block its accessibility and/or affect its conformation.
- Klíčová slova
- 14-3-3 protein, apoptosis signal-regulating kinase 1 (ASK1), fluorescence, nuclear magnetic resonance (NMR), protein cross-linking, small-angle x-ray scattering (SAXS),
- MeSH
- difrakce rentgenového záření MeSH
- fosforylace MeSH
- katalytická doména MeSH
- lidé MeSH
- maloúhlový rozptyl MeSH
- MAP kinasa-kinasa-kinasa 5 antagonisté a inhibitory chemie genetika metabolismus MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- proteiny 14-3-3 chemie genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- MAP kinasa-kinasa-kinasa 5 MeSH
- MAP3K5 protein, human MeSH Prohlížeč
- proteiny 14-3-3 MeSH
- YWHAE protein, human MeSH Prohlížeč
Ca2+ /CaM-dependent protein kinase kinases 1 and 2 (CaMKK1 and CaMKK2) phosphorylate and enhance the catalytic activity of downstream kinases CaMKI, CaMKIV, and protein kinase B. Accordingly, CaMKK1 and CaMKK2 regulate key physiological and pathological processes, such as tumorigenesis, neuronal morphogenesis, synaptic plasticity, transcription factor activation, and cellular energy homeostasis, and promote cell survival. Both CaMKKs are partly inhibited by phosphorylation, which in turn triggers adaptor and scaffolding protein 14-3-3 binding. However, 14-3-3 binding only significantly affects CaMKK1 function. CaMKK2 activity remains almost unchanged after complex formation for reasons still unclear. Here, we aim at structurally characterizing CaMKK1:14-3-3 and CaMKK2:14-3-3 complexes by SAXS, H/D exchange coupled to MS, and fluorescence spectroscopy. The results revealed that complex formation suppresses the interaction of both phosphorylated CaMKKs with Ca2+ /CaM and affects the structure of their kinase domains and autoinhibitory segments. But these effects are much stronger on CaMKK1 than on CaMKK2 because the CaMKK1:14-3-3γ complex has a more compact and rigid structure in which the active site of the kinase domain directly interacts with the last two C-terminal helices of the 14-3-3γ protein, thereby inhibiting CaMKK1. In contrast, the CaMKK2:14-3-3 complex has a looser and more flexible structure, so 14-3-3 binding only negligibly affects the catalytic activity of CaMKK2. Therefore, Ca2+ /CaM binding suppression and the interaction of the kinase active site of CaMKK1 with the last two C-terminal helices of 14-3-3γ protein provide the structural basis for 14-3-3-mediated CaMKK1 inhibition.
- Klíčová slova
- 14-3-3 proteins, CaMKK, SAXS, calcium/calmodulin-dependent protein kinase, fluorescence spectroscopy, hydrogen/deuterium exchange coupled to MS, protein-protein interaction,
- MeSH
- difrakce rentgenového záření MeSH
- fosforylace MeSH
- katalytická doména MeSH
- kinasa proteinkinasy závislé na vápníku a kalmodulinu * chemie metabolismus MeSH
- maloúhlový rozptyl MeSH
- proteiny 14-3-3 * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- kinasa proteinkinasy závislé na vápníku a kalmodulinu * MeSH
- proteiny 14-3-3 * MeSH
The gene pkwA coding for a typical WD-repeat protein was found in the chromosome of the bacterium Thermomonospora curvata CCM 3352. Until now WD-repeat proteins were through to be confined to eukaryotes.
- MeSH
- Actinomycetales enzymologie genetika MeSH
- bakteriální geny MeSH
- bakteriální proteiny genetika MeSH
- molekulární sekvence - údaje MeSH
- protein-serin-threoninkinasy genetika MeSH
- repetitivní sekvence nukleových kyselin * MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- sekvenční homologie aminokyselin MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
- Názvy látek
- bakteriální proteiny MeSH
- PkwA protein, Thermomonospora curvata MeSH Prohlížeč
- protein-serin-threoninkinasy MeSH
The fine tuning of hormone (e.g., auxin and gibberellin) levels and hormone signaling is required for maintaining normal embryogenesis. Embryo polarity, for example, is ensured by the directional movement of auxin that is controlled by various types of auxin transporters. Here, we present pieces of evidence for the auxin-gibberellic acid (GA) hormonal crosstalk during embryo development and the regulatory role of the Arabidopsis thaliana Calcium-Dependent Protein Kinase-Related Kinase 5 (AtCRK5) in this regard. It is pointed out that the embryogenesis of the Atcrk5-1 mutant is delayed in comparison to the wild type. This delay is accompanied with a decrease in the levels of GA and auxin, as well as the abundance of the polar auxin transport (PAT) proteins PIN1, PIN4, and PIN7 in the mutant embryos. We have previously showed that AtCRK5 can regulate the PIN2 and PIN3 proteins either directly by phosphorylation or indirectly affecting the GA level during the root gravitropic and hypocotyl hook bending responses. In this manuscript, we provide evidence that the AtCRK5 protein kinase can in vitro phosphorylate the hydrophilic loops of additional PIN proteins that are important for embryogenesis. We propose that AtCRK5 can govern embryo development in Arabidopsis through the fine tuning of auxin-GA level and the accumulation of certain polar auxin transport proteins.
- Klíčová slova
- Arabidopsis thaliana, Calcium-Dependent Protein Kinase-Related Kinase (CRK), GA3, auxin gradient, embryogenesis, polar auxin transport (PAT) proteins,
- MeSH
- Arabidopsis růst a vývoj metabolismus MeSH
- ELISA MeSH
- gibereliny analýza metabolismus MeSH
- klíčení * MeSH
- kyseliny indoloctové metabolismus MeSH
- membránové transportní proteiny genetika metabolismus MeSH
- protein-serin-threoninkinasy metabolismus MeSH
- proteiny huseníčku genetika metabolismus MeSH
- receptory buněčného povrchu metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- semena rostlinná anatomie a histologie růst a vývoj metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- CRK5 protein, Arabidopsis MeSH Prohlížeč
- gibberellic acid MeSH Prohlížeč
- gibereliny MeSH
- kyseliny indoloctové MeSH
- membránové transportní proteiny MeSH
- protein-serin-threoninkinasy MeSH
- proteiny huseníčku MeSH
- receptory buněčného povrchu MeSH
Apoptosis signal-regulating kinase 1 (ASK1, also known as MAP3K5), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, activates the p38 mitogen-activated protein kinase and the c-Jun N-terminal kinase (JNK) signaling cascades in response to various stressors. ASK1 activity is tightly regulated through phosphorylation and interaction with various binding partners. However, the mechanistic details underlying the ASK1 regulation are still not fully understood. This review focuses on recent advances in structural studies of protein kinase ASK1 and on the insights they provide into its mechanism of regulation. In addition, we also discuss protein-protein interactions between ASK1 and its binding partners thioredoxin (TRX) and 14-3-3 protein.
- MeSH
- biologické modely MeSH
- fosforylace MeSH
- lidé MeSH
- MAP kinasa-kinasa-kinasa 5 genetika metabolismus MeSH
- mitogenem aktivované proteinkinasy p38 genetika metabolismus MeSH
- proteiny 14-3-3 genetika metabolismus MeSH
- thioredoxiny genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- MAP kinasa-kinasa-kinasa 5 MeSH
- mitogenem aktivované proteinkinasy p38 MeSH
- proteiny 14-3-3 MeSH
- thioredoxiny MeSH
A 4.2-kb SphI-BamHI fragment of chromosomal DNA from Streptomyces granaticolor was cloned and shown to encode a protein with significant sequence similarity to the eukaryotic protein serine/threonine kinases. It consists of 701 amino acids and in the N-terminal part contains all conserved catalytic domains of protein kinases. The C-terminal domain of Pkg2 contains seven tandem repeats of 11 or 12 amino acids with similarity to the tryptophan-docking motif known to stabilize a symmetrical three-dimensional structure called a propeller structure. The pkg2 gene was overexpressed in Escherichia coli, and the gene product (Pkg2) has been found to be autophosphorylated at serine and threonine residues. The N- and C-terminal parts of Pkg2 are separated with a hydrophobic stretch of 21 amino acids which translocated a PhoA fusion protein into the periplasm. Thus, Pkg2 is the first transmembrane protein serine/threonine kinase described for streptomycetes. Replacement of the pkg2 gene by the spectinomycin resistance gene resulted in changes in the morphology of aerial hyphae.
- MeSH
- bakteriální geny MeSH
- DNA bakterií genetika MeSH
- DNA primery genetika MeSH
- Escherichia coli genetika MeSH
- exprese genu MeSH
- fenotyp MeSH
- fosforylace MeSH
- klonování DNA MeSH
- mikroskopie elektronová rastrovací MeSH
- molekulární sekvence - údaje MeSH
- mutace MeSH
- protein-serin-threoninkinasy chemie genetika metabolismus MeSH
- rekombinantní fúzní proteiny chemie genetika metabolismus MeSH
- restrikční mapování MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- sekvenční homologie aminokyselin MeSH
- Streptomyces enzymologie genetika ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA bakterií MeSH
- DNA primery MeSH
- protein-serin-threoninkinasy MeSH
- rekombinantní fúzní proteiny MeSH
Searching the genome sequence of Streptococcus pneumoniae revealed the presence of a single Ser/Thr protein kinase gene stkP linked to protein phosphatase phpP. Biochemical studies performed with recombinant StkP suggest that this protein is a functional eukaryotic-type Ser/Thr protein kinase. In vitro kinase assays and Western blots of S. pneumoniae subcellular fractions revealed that StkP is a membrane protein. PhpP is a soluble protein with manganese-dependent phosphatase activity in vitro against a synthetic substrate RRA(pT)VA. Mutations in the invariant aspartate residues implicated in the metal binding completely abolished PhpP activity. Autophosphorylated form of StkP was shown to be a substrate for PhpP. These results suggest that StkP and PhpP could operate as a functional pair in vivo. Analysis of phosphoproteome maps of both wild-type and stkP null mutant strains labeled in vivo and subsequent phosphoprotein identification by peptide mass fingerprinting revealed two possible substrates for StkP. The evidence is presented that StkP can phosphorylate in vitro phosphoglucosamine mutase GlmM which catalyzes the first step in the biosynthetic pathway leading to the formation of UDP-N-acetylglucosamine, an essential common precursor to cell envelope components.
- MeSH
- 2D gelová elektroforéza MeSH
- fosfoglukomutasa metabolismus MeSH
- fosforylace MeSH
- hmotnostní spektrometrie MeSH
- mangan metabolismus MeSH
- plazmidy MeSH
- protein-serin-threoninkinasy chemie metabolismus MeSH
- proteinfosfatasa 2C MeSH
- proteinfosfatasy metabolismus MeSH
- proteom MeSH
- rekombinantní fúzní proteiny metabolismus MeSH
- Streptococcus pneumoniae enzymologie MeSH
- subcelulární frakce MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- fosfoglukomutasa MeSH
- mangan MeSH
- phosphoglucosamine mutase MeSH Prohlížeč
- protein-serin-threoninkinasy MeSH
- proteinfosfatasa 2C MeSH
- proteinfosfatasy MeSH
- proteom MeSH
- rekombinantní fúzní proteiny MeSH